JOURNAL Structure of the entire cytoplasmic portion of a...

Structure of the entire cytoplasmic portionof a sensor histidine-kinase protein

Alberto Marina1,2, Carey D Waldburger3,4

and Wayne A Hendrickson1,*1Howard Hughes Medical Institute, Department of Biochemistryand Molecular Biophysics, Columbia University, New York, NY, USA,2Macromolecular Crystallography Unit, Instituto de Biomedicinade Valencia (CSIC), Valencia, Spain and 3Department of Microbiology,Columbia University, New York, NY, USA

The large majority of histidine kinases (HKs) are multi-

functional enzymes having autokinase, phosphotransfer

and phosphatase activities, and most of these are trans-

membrane sensor proteins. Sensor HKs possess conserved

cytoplasmic phosphorylation and ATP-binding kinase

domains. The different enzymatic activities require parti-

cipation by one or both of these domains, implying the

need for different conformational states. The catalytic

domains are linked to the membrane through a coiled-

coil segment that sometimes includes other domains. We

describe here the first crystal structure of the complete

cytoplasmic region of a sensor HK, one from the thermo-

phile Thermotoga maritima in complex with ADPbN at

1.9 A resolution. The structure reveals previously uniden-

tified functions for several conserved residues and reveals

the relative disposition of domains in a state seemingly

poised for phosphotransfer. The structure thereby inspires

hypotheses for the mechanisms of autophosphorylation,

phosphotransfer and response-regulator dephosphoryla-

tion, and for signal transduction through the coiled-coil

segment. Mutational tests support the functional relevance

of interdomain contacts.

The EMBO Journal (2005) 24, 4247–4259. doi:10.1038/

sj.emboj.7600886; Published online 1 December 2005

Subject Categories: signal transduction; structural biology

Keywords: crystal structure; PhoQ; phosphotransfer;

selenomethionyl MAD; two-component systems

Introduction

Nearly all living cells use phosphorylation-mediated signal

transduction mechanisms in responding to metabolic, envir-

onmental and cell-cycle stimuli. ‘Two-component’ regulatory

systems involving His-Asp phosphorelays predominate for

signal transduction in prokaryotes and are commonplace in

fungi and plants (reviewed by Stock et al, 2000). The para-

digmatic two-component system consists of two basic protein

units: a sensor histidine kinase (HK) and a response regulator

(RR). The former acts as the signal receptor and possesses an

autokinase activity that promotes phosphorylation of a histi-

dine residue in a conserved domain. The phosphoryl group

is then transferred to an aspartate residue of the RR (usually

a transcription factor), triggering the cellular response. The

response is proportional to the degree of RR phosphorylation,

which depends not only on the efficiency of the autokinase

and transfer reactions but also in many cases on an intrinsic

autophosphatase activity in the RR and/or destabilization of

the aspartyl phosphate bond by the cognate HK (termed

regulated phosphatase activity). Signals typically mediate

responses by influencing the HK autokinase and/or phospha-

tase activity (Russo and Silhavy, 1993).

The large majority of HKs, labeled class I HKs (Bilwes et al,

1999), are homodimeric membrane proteins in which

each subunit contains a short amino-terminal cytoplasmic

segment followed by a transmembrane a helix (TM1) and

an extracellular (or periplasmic) sensing domain that is

connected via a second membrane-spanning a helix (TM2)

to a carboxy-terminal cytoplasmic kinase domain (Stock et al,

2000). The extracellular sensing domains are variable in

sequence, reflecting the wide range of environmental signals

to which HKs respond. Conversely, the cytoplasmic portion

typically includes a conserved catalytic core of approximately

250 residues, which contains a set of characteristic sequence

motifs, labeled the H, N, G1, F and G2 boxes (Parkinson

and Kofoid, 1992). This core portion of class I HKs can be

dissected into two distinct functional domains: an N-terminal

dimerization and histidine phosphotransfer (DHp) domain

and a C-terminal catalytic and ATP-binding (CA) domain.

The DHp domain, which contains the autophosphorylation

site (H box), forms a stable dimer and can be phosphorylated

in the presence of ATP by the CA domain (Stock et al, 2000).

The isolated CA domain is monomeric and encompasses the

conserved N, G1, F, and G2 boxes.

The segment that connects TM2 to the catalytic core in

class I HKs is variable in length and sequence, but it typically

includes a common structural element called the HAMP

(histidine kinase, adenylyl cyclase, methyl-accepting chemo-

taxis proteins and phosphatase) or P-type linker (Aravind

and Ponting, 1999; Williams and Stewart, 1999). HAMP

linkers are variable in length (40–180 residues) and have

a predicted topology of two amphipathic helices separated

by a loop region. They have been hypothesized to transmit

signals between the external input domain and the cyto-

plasmic output module (Fabret et al, 1999; Williams and

Stewart, 1999).

Atomic structures have been reported for the isolated DHp

and CA domains of some HK sensors. Structures of both

dissected domains have been determined by NMR spectro-

scopy for the osmosensor EnvZ from Escherichia coli (Tanaka

et al, 1998; Tomomori et al, 1999), and CA domain structures

have been determined by X-ray crystallography for the

Thermotoga maritima CheA, E. coli PhoQ and NtrB CAReceived: 6 May 2005; accepted: 3 November 2005; published online:1 December 2005

*Corresponding author. Howard Hughes Medical Institute, Departmentof Biochemistry and Molecular Biophysics, Columbia University, NewYork, NY 10032, USA. Tel.: þ 1 212 305 3456; Fax: þ 1 212 305 7379;E-mail: [email protected] address: Department of Biology, William Paterson University,Wayne, NJ 07474, USA

The EMBO Journal (2005) 24, 4247–4259 | & 2005 European Molecular Biology Organization | All Rights Reserved 0261-4189/05

www.embojournal.org

&2005 European Molecular Biology Organization The EMBO Journal VOL 24 | NO 24 | 2005

EMBO

THE

EMBOJOURNAL

THE

EMBOJOURNAL

4247

domains (Bilwes et al, 1999; Marina et al, 2001; Song et al,

2004). The EnvZ DHp domain consists of two a helices that

dimerize to form a four-helix bundle in which the histidine

phospho-acceptors protrude from helices into the solvent

(Tomomori et al, 1999). The four CA domains assume a

mixed a/b sandwich fold made from five b strands and

three a helices. These kinase domains are structurally related

to the ATP-binding domains of the GHL ATPase family

(GyraseB, Hsp90 and MutL); thereby these ATPase become

the GHKL superfamily (Dutta and Inouye, 2000).

The three enzymatic activities (autokinase, phosphotrans-

fer and phosphatase) associated with the cytoplasmic region

of the HK each require the participation of one or both of the

DHp and CA domains (Tanaka et al, 1991; Hsing et al, 1998),

suggesting that these domains can exist in different confor-

mational states with respect to one another. Structural char-

acterization of these signaling states has been thwarted in the

dissection approach. Here we have analyzed an intact cyto-

plasmic domain from a HK sensor protein, that of T. maritima

TM0853. The resulting structure inspires testable hypotheses

about the mechanism of signal transduction in HKs.

Results

Characterization and structure determination

In a survey aimed at expressing the cytoplasmic portions

of HK sensors, we succeeded to clone, express and purify

a fragment of a protein from T. maritima (ORF TM0853). This

is a putative HK sensor by virtue of sequence similarities that

are especially striking in the catalytic domain (Figure 1).

Much of the full-length protein partitioned into the soluble

fraction when expressed in E. coli, but the membrane-

associated fraction increased with temperature consistent

with membrane localization in the natural thermophilic

Figure 1 Sequence alignment of TM0853, EnvZ and PhoQ HKs. The three amino-acid sequences are aligned based on their structures. b sheetsare shown as blue arrows and a helices as yellow filled boxes. Transmembrane regions predicted by the DAS program (Cserzo et al, 1997) areshown as purple intermediate shading and coiled-coil motifs predicted by LEARNCOIL program (Singh et al, 1998) are enclosed in blue boxesshowing the helical position from a to g. Disordered regions are enclosed in red boxes. Residues identical in all three sequences are colored inred. The solvent accessibility of the HK853–CD is indicated for each residue by an open circle if the fraction solvent accessibility is 40.4, a half-filled circle if it is 0.1–0.4 and a filled circle if it is o0.1. Residues that interact with the ADPbN and the sulfate ion in HK853–CD are indicatedby green and red diamonds, respectively.

Cytoplasmic structure of a sensor histidine kinaseA Marina et al

The EMBO Journal VOL 24 | NO 24 | 2005 &2005 European Molecular Biology Organization4248

host (Table I). We defined its cytoplasmic portion to comprise

residues 233–489 and produced the corresponding fragment

(HK853-CD). The purified protein is dimeric, as estimated by

gel filtration chromatography and crosslinking assays (data not

shown). HK853-CD was shown to possess an ATP-dependent

autokinase activity in vitro (Figure 2A) and to support phos-

photransfer to both PhoP and OmpR RRs (Figure 2B). Crystals

of HK853-CD grown in the presence of the inert ATP analog

AMPPNP diffracted beyond 2 A Bragg spacings. Despite the

amino-acid sequence similarity (Figure 1), efforts to solve the

HK853-CD structure by molecular replacement based on mod-

els of either the PhoQ or EnvZ catalytic subdomains were

unsuccessful. Consequently, the structure was determined from

a selenomethionine-substituted protein by the MAD method.

An atomic model of HK853-CD was built and refined

to 1.9 A resolution. The electron densities for the nine

C-terminal residues and internal-loop residues 433–441

were extremely weak or absent, and these regions were

presumed to be disordered. The refined structure contains

240 residues, a hydrolyzed AMPPNP molecule, one sulfate

ion and 179 water molecules, and it has good stereochemistry

with no Ramachandran outliers (Table II).

Overall structure

There is one HK853-CD subunit in the asymmetric unit of

the crystal, and it exploits a two-fold symmetry axis of the

lattice to generate a homodimer, as expected from our solution

studies and results on homologous systems (Yang and Inouye,

1991; Ninfa et al, 1993). Each protomeric subunit consists of

two distinct domains, an N-terminal helical hairpin domain

and a C-terminal a/b domain, which are connected by a short

linker (residues 318–322) (Figure 3). The dimer interface is

exclusively between helical-hairpin domains and the diad axis

runs parallel with the helices such that the N-termini are

adjacent, as if poised to emanate from the membrane.

The helical-hairpin domain comprises residues 232–317

and has its two antiparallel helices connected by a nine-

residue turn (residues 279–287). The first helix extends for

about 75 A from the N-terminus (232) to residue 278, but it

has a pronounced kink induced by Pro265 such that we

designate two parts; helix a1a includes the His260 phosphor-

ylation site and helix a1b makes helix–bundle contacts with

helices a2 and a20, the symmetry mate. Helix a2 (residues

288–317) is shorter (B50 A).

The C-terminal domain (residues 323–489) assumes an

a/b sandwich fold: one layer comprises a mixed five-strands

b sheet (bB, bD–bG), which is nearly orthogonal to the

helical-hairpin structure, and the other layer consists

of three a helices (a3–a5). In addition, this domain contains

a pair of short antiparallel b strands (bA and bC) and one

disulfide bridge (Cys330–Cys359) linking the N-terminal

segment of a3 (just following bA) with bC.

Dimeric association of helical-hairpin domains

The dimer of helical hairpin domains has two parts, a coiled-

coil portion composed of the 22 N-terminal residues of helix

a1a and a four-helix bundle portion composed of the rest

(Figures 3 and 4). C-terminal segments of the a1 helices each

interact with both a2 helices in an antiparallel manner with

a left-handed twist of about 251, the most favored assembly

for a four-helix bundle (Chou et al, 1988). The four-helix

bundle portion corresponds to the DHp domain and the

coiled-coil extension includes the HAMP or P-type linker.

His 260, the H-box histidine and presumed site of phosphor-

ylation, is on the surface of this four-helix bundle. There is

a large interface of association, burying 2100 A2 of solvent-

accessible surface area from each protomer. The majority

of this interface is in the four-helix bundle (1500 A2), but the

coiled-coil interface is also substantial (600 A2).

Several hydrophobic residues at the four-helix bundle

interface are conserved among HKs. This interface also

Table I Expression of full-length HK853 in E. coli

T (1C) Distribution of HK853between cellular

compartments (%)

HK853 portion of totalprotein within each

cellular compartment (%)

Membrane Soluble Membrane Soluble

20 5.070.8 95.070.8 31.5716.1 32.076.425 8.571.2 91.571.2 57.776.5 33.074.530 14.672.7 85.472.7 60.975.5 26.373.837 25.977.7 74.177.7 70.475.9 26.1710.3

Results are averages of two independent experiments, each of whichwas quantified twice (two independent gels). Possible inclusionbodies were eliminated before analysis.

0 15′′ 1′ 2.5′ 10′

HK853~P

β-G

alac

tosi

das

e ac

tivi

ty

pHK853 pEnvZ pPhoQ pBR3220

500

1000

1500

∆PhoQ∆EnvZ

A

B

Figure 2 In vitro and in vivo HK853 activity. (A) Time course ofin vitro autophosphorylation of HK853–CD with [g-32P]ATP. In all,1–2 mM of HK853–CD was incubated in reaction buffer and sampleswere removed at indicated time points, reaction stopped by additionof SDS–PAGE sample buffer, subjected to gel electrophoresis,and phosphorylated protein was visualized by phosphorimaging.(B) Activation of the PhoQ–PhoP and EnvZ–OmpR HK–RR systems.E. coli reporter strains containing a PhoP-activated lacZ, but devoidof PhoQ (DPhoQ), or an OmpR-activated lacZ, but devoid of EnvZ(DEnvZ), were transformed with the pBR322-derived plasmidspHK853, pEnvZ, pPhoQ or pBR322 (expressing the respective full-length sensor kinases or a negative control). Response was assayedby b-galactosidase activity. The low activation by PhoQ was due torepressing divalent cations in culture media.


&2005 European Molecular Biology Organization The EMBO Journal VOL 24 | NO 24 | 2005 4249

includes hydrophilic interactions, notably two hydrogen bonds

(Thr252–Glu3160 and Arg263–Asn3070) and one salt bridge

(Lys270–Glu3030), although of these only Arg263 is conserva-

tive. At the apex of the bundle, the connection between a1b

and a2 helices is rather extended and consists of alternately

exposed polar and buried hydrophobic residues. This segment

is intrinsically flexible, as judged by elevated B factors. The a2

helices splay apart C-terminally (from 11 A along the bundle to

18 A at the open end), and the a1a helices emerge from

between the separated a2 helices to meet in the coiled coil.

The transition from antiparallel bundle to parallel coiled-

coil interactions generates a small cavity filled with water

molecules (Figure 4B). Ile255 (from a1 helices), and Leu309

and Phe312 (from a2 helices) form the bottom portion of this

cavity and are conserved in class I HKs. In contrast, the

middle and upper portions are composed of polar (Thr252)

and charged (Lys251 and Glu316) residues that interact with

waters of the cavity. A hydrogen bond between carboxylate

Od1 atoms of Asp248 and its symmetry mate Asp2480 (one

presumably protonated) closes the top of the cavity and

initiates the N-terminal coiled-coil interactions. The Od2

atoms of Asp248 and Asp2480 are both hydrogen bonded to

the same water molecule of the cavity, which makes addi-

tional hydrogen bounds with other cavity waters. Side chains

of Leu241 and Leu244 pack against their symmetry-related

residues to form part of the hydrophobic core of the coiled-

coil N-terminal segment.

The four-helix bundle domain of HK853 differs from the

NMR-derived structure of the isolated DHp domain from

E. coli EnvZ (Tomomori et al, 1999) in two significant

respects. Firstly, the twist angle of the HK853 four-helix

bundle (B251) is higher than that of EnvZ (B101), which

is unusually parallel for this topological class (Dutta et al,

1999). Secondly, the connections between hairpin helices are

crossed in the two models, that is, a2 and a20 are inter-

changed. This is a surprising difference for members of the

same sequence family, especially as the four-helix bundle

topology of the more distantly related Bacillus subtilis Spo0B

histidine phosphotransferase (Varughese et al, 1998) is the

same as that observed for HK853 (Figure 4A). The connecting

segment between helices was reported as unstructured in

EnvZ, raising concern about the linkage geometry, but the

use of mixtures of labeled and unlabeled EnvZ protein in the

NMR analysis should have distinguished interchain from

intrachain interactions. The helical-hairpin connector also

has elevated atomic mobility in the HK853 structure, but

the path of connecting electron density is unambiguous. This

topological distinction, if indeed real, necessarily has

mechanistic implications. The HK853 structure is consistent

with trans-phosphorylation, as is observed (Yang and

Inouye, 1991; Ninfa et al 1993; Qin et al, 2000), whereas

the alternative would seem to be only consistent with phos-

phorylation on the same chain unless the DHp to CA linkage

is radically different in EnvZ. It may also be that the truncated

Table II Diffraction and structure determination statistics

HK853-CD Selenomethionyl I370M/V373M HK853–CD

Native Selow Seedge Sepeak Sehigh

Diffraction dataWavelength (A) 0.9678 0.9918 0.9794 0.9788 0.9678Spacing limit (A) 1.9 2.1 2.1 2.1 2.1Unique reflections 21 892 16 406 16 405 16 416 16 434Rmerge (%)a 6.1 (26.8) 4.6 4.5 4.6 4.8I/s 13.8 (2.7) 11.9 12.3 12.1 11.3Completeness (%) 98.0 (99.7) 97.7 95.5 96.8 97.7Redundancy 13.4 (5.2) 7.3 6.2 6.9 7.3

MAD phasingPhasing powerb (DDl/D7h) 2.64/2.09 3.04/3.13 0.91/2.46Rcullis

c (DDl/D7h) 0.51/0.79 0.48/0.58 0.68/0.71Overall FOM (acentric/centric)d 0.68/0.50

RefinementBragg spacings (A) 20–1.9Re/Rfree

f (%) 24.7/27.5 (27.9/32.0)Number of protein atoms 1960Number of solvent atoms 179Number of non-protein atoms 33Average B factor (A2) 35.6R.m.s. bonds (A), angles (deg) 0.011/1.7Ramachandran analysisg

Favored/outlier (%) 97.7/0.0

Values in parentheses refer to the highest resolution shell (2.0–1.9).aRmerge¼

P|I�/IS|/

PI, where I is the observed intensity and /IS the average intensity.

bPhasing power¼ root-mean-square (Fh/E), where Fh¼heavy atom structure factor amplitude and E¼ residual lack of closure error. DDl is fordispersive differences relative to Selow. D7h is for Bijvoet differences.cRcullis¼

P||Fh(obs)-|Fh(calc)||/

P|Fh(obs)|, where Fh(obs) and Fh(calc) are the observed and calculated heavy atoms structural factor

amplitudes, respectively.dFigure of merit¼ |F(hkl)best|/F(hkl).eR¼

P||Fo|�|Fc||/

P|Fo|.

fRfree is calculated as R, but on 5.2% of all reflections that are never used in crystallographic refinement.gAnalysis from http://kinemage.biochem.duke.edu.



EnvZ DHp domain has an unnatural conformation in the

absence of its kinase and coiled-coil neighbors.

Nucleotide binding in the kinase domain

The CA domain in HK853-CD is similar in structure to the

corresponding kinase domains isolated from PhoQ (Marina

et al, 2001), CheA (Bilwes et al, 1999), EnvZ (Tanaka et al,

1998) and NtrB (Song et al, 2004). Superimpositions of 125

Ca atoms of the PhoQ, NtrB and CheA domains with CA of

HK853 give r.m.s. deviations of 1.4, 1.3 and 1.6 A2, respec-

tively. Although the protein was crystallized in presence of

the inert ATP analog AMPPNP and MgCl2, no electron density

was observed for the g phosphate or for Mg2þ ions. We

subsequently discovered that the AMPPNP nucleotide was

hydrolyzed in the crystallization buffer (Na cacodylate (pH

6.5)þ LiSO4) into a distinct product similar to ADP, which we

identify as ADPbN (ADP-NH2 as designed by Yount et al,

1971) (see Materials and methods). Surprisingly, ATP is

stable under these conditions. In PhoQ and CheA structures,

the Mg2þ ion bridges the three nucleotide phosphates

(Bilwes et al, 2001; Marina et al, 2001), suggesting that the

absence of the Mg2þ ion in the HK853-CD structure could be

due to the loss of g-phosphate interaction. The nucleotide in

the ADP–CheA complex (Bilwes et al, 2001), which also lacks

the metal ion, is positioned similarly to ADPbN in HK853.

The segment that joins the conserved F and G2 boxes

of HKs has the flexibility to adopt different conformations. In

PhoQ and CheA it covers the nucleotide (Bilwes et al, 2001;

Marina et al, 2001); hence, it is called the ATP lid. In HK853-

CD, residues 433–441 of the ATP lid are disordered, suggest-

ing high mobility in absence of the g phosphate and the

Mg2þ . Similarly, the ATP lid was not observable in CheA and

Figure 3 Molecular structure of the cytoplasmic portion of TM0853. (A) Ribbon representation of the crystallographic dimer of HK853–CD,including ADPbN. The a helices are labeled a1–a5, and colored gold (subunit A) and green (subunit B), b strands are labeled bA–bF,and colored blue (subunit A) and red (subunit B). The positions of N and C termini are labeled in subunit B. The ADPbN molecule and thephospho-acceptor residue (His260) are shown in ball-and-stick representation. The membrane would be located on the top of N-terminalresidues. (B) Stereo Ca trace of the gold and blue protomer. Every tenth Ca is indicated as a sphere and numbered. The ADPbN molecule, theHis260 and the sulfate ion coordinated with His260 are drawn in a gray ball-and-stick representation. The orientation is as in panel (A).



NtrB structures solved with empty nucleotide sites (Bilwes

et al, 1999; Song et al, 2004). The ATP-lid segment of HK853

is anchored at each end by hydrophobic residues, the F-box

namesake Phe425 and conservative Leu446, which interact

with one another and with other hydrophobic residues

(Ile424 and Ile460 in HK853) as in PhoQ and CheA counter-

parts (Marina et al, 2001). This supports the generality of this

hydrophobic patch motif in ATP-lid attachment.

We have previously described two major groups of HKs,

typified by PhoQ and CheA, based on nucleotide-binding

geometry and mechanistic roles of key residues (Marina

et al, 2001). The HK853 kinase belongs to the predominant

PhoQ group, which also includes EnvZ (Figure 1), and

nucleotide-binding residues in HK853 are disposed as they

are in PhoQ (Figure 5). Structurally, an aromatic residue

(Tyr393 in PhoQ, Tyr384 in HK853) is sandwiched between

the adenine base, with which it stacks, and aliphatic por-

tions of a basic residue (Lys392 in PhoQ, Lys 383 in HK853).

Both functional groups make hydrogen bonds with nucleo-

tide phosphates (g in PhoQ, b in HK853). In addition, this

sequence group has a conserved arginine or glutamine in the

ATP lid, and both in PhoQ and in HK853 these residues

interact with nucleotide b phosphates, suggesting a catalytic

role for Arg430 of HK853 analogous to that found for Arg434

of PhoQ. Overall, the HK853 kinase structure supports our

previous suggestions on histidine-kinase classification and

catalytic mechanism.

Environment of the histidine phosphoacceptor site

His260 in HK853 corresponds to the absolutely conserved

histidine, at which phosphorylation has been established

to occur in representative HKs. Its side chain is fully exposed

on helix a1 near the a1a–a1b kink provoked by Pro265

(Figure 4). An electron-dense feature, consistent with a sulfate

ion coming from the 1.3 M SO42� present in the crystallization

mixture, is located nearby, with oxygen atom O1 of this

sulfate ion 2.6 A from Ne of His260. The Ne atom on the

imidazole ring is a more stable phosphorylation site than Nd(Hultquist et al, 1996), and NMR studies confirmed it as the

phosphorylation site in CheA (Zhou and Dahlquist, 1997).

Based on these features and chemical similarities between

sulfate and phosphate, it seems reasonable to conclude that

the histidyl sulfate interaction may be mimicking phosphor-

ylation of the histidine. This His260-associated sulfate ion

also interacts with Arg3170, Arg3140 and Ser3190 of the neigh-

boring protomer in the dimer (Figure 6), making hydrogen

bonds to the side chains of Arg3170 (O1–NZ2 at 3.0 A and O2–

NZ1 at 2.9 A), Arg3140 (O1–Ne at 3.0 A) and Ser3190 (O3–Ogat 2.6 A). These three residues reside in a weakly conserved

motif, termed X region (Hsing et al, 1998), which comprises

the C-terminal end of helix a2 and the interdomain linker in

the HK853-CD structure. The X region has been implicated in

regulating phosphatase activity (see Discussion).

Interactions between the two cytoplasmic domains

This structure provides the first picture of interdomain con-

tacts between the catalytic and DHp domains of a histidine-

kinase sensor. The domains are connected by an extended

linker (residues 318–322) in this state of the molecule, but

a substantial interface between domains (1250 A2) is never-

theless buried from solvent exposure. Contacts are formed

principally from conserved hydrophobic residues and they

E237

S238

L241

E240K245L244

D248

K251T252

I255

H260

L309

F312

E316

EnvZ HK853Spo0B

α1

α2

Coiled coil

Four-helixbundle

A B

Figure 4 Comparison of DHp domains of EnvZ HK, Spo0B phosphotransferase and TM0853 HK. (A) Ribbon diagrams of the three DHpdomains in their dimeric forms (protomers colored in green and gold). The DHp domains have been oriented with the plane containing thephospho-accepting histidines, which are shown in stick representation, and the principal helix axes parallel to the page. (B) Detail of the HK853coiled-coil motif. Residues interacting in this motif are shown as stick representation and labeled on one protomer. Solvent molecules(magenta) are shown in the cavity generated at the juncture between the coiled coil of a1a helices and the four-helix bundle.



are exclusively within a promoter (Figure 6). The DHp

domain contributes 525 A2 to the buried surface area, invol-

ving residues from helices a1a and a2 helices at the open

end of the helical hairpin; the catalytic domain buries 475 A2

from the a3 helix, the conserved G2 box (N-terminus of helix

a4) and the conserved F box; and residues from the linker

segment add the remaining 250 A2.

The interaction interface can be divided into two patches

grouped around each helix of the DHp domain. The a2 helix

interacts with the a3 and a4 (G2 box) helices of the cata-

lytic domain and with the connecting loop. In detail, there is

a cluster of hydrophobic side chains, contributed by Leu315

and Phe312 in a2 (DHp), Leu320 and Ile322 (linker), and

Leu444, Ala447 and Ile448 in a4 (CA), that is flanked on each

side by hydrophilic residues exposing the aliphatic portion of

their side chains to this cluster. Those at one side, Arg369

and Gln372 from a4 and Asp311 in a2, are hydrogen bonded

together in a unique triple residue interaction. The second

region is characterized by the projection of one of the

conserved F box phenylalanines (Phe428) towards the a1

helix, where it is accommodated in a hydrophobic pocket

formed by Ile247, Met250, Phe254 and the aliphatic

portion of the Lys251 side chain. The two buried areas are

connected through an interaction between Phe254 in a1 and

Phe312 in a2.

Mutational tests for functional relevance of interdomain

contacts

The structure invites the hypothesis that contacts seen

between DHp and CA domains may support a labile associa-

tion, under control of the sensor domain, to be released

for autophosphorylation and maintained for phosphatase

and phosphotransferase activities. To examine this hypoth-

esis, we designed a series of 15 mutant variants, incorporat-

ing mutations at seven sites, and tested them for functional

relevance in our autokinase assay. These mutations are in

three classes: changes at interfacial hydrophobic residues,

introductions of candidates for disulfide formation and pro-

line substitutions in the interdomain linker segment. Figure 7

compares the kinetics of autophosphorylation with wild-type

activity for several of these variants, and Supplementary

Table SII records the initial rates for all. Most of the mutations

affect autokinase activity significantly, usually in the initial

rate of phosphorylation and also, often differentially, in the

achieved or projected equilibrium level. Equilibrium levels

may reflect differences in rates of counteracting intrinsic

dephosphorylation, so we concentrate our analysis on

the initial rates.

The mutational analysis provides compelling evidence for

the importance of this interface in controlling autokinase

activity. Activity is sensitive to mutation at all tested inter-

facial hydrophobic residues (Supplementary Table SII) and

most strikingly so at Ile448 (Figure 7A); I448A had a six-fold

rate increase, but I448W had negligible activity despite good

solution properties. Activities for variants L315W and F428E

were also strongly affected (B3-fold increases). The double

cysteine mutations, F312C/L444C and L315C/L444C, further

corroborate the functional significance of the interface

(Figure 7B). There is no activity under conditions conducive

to disulfide coupling of the domains, but activity increases

to rates consistent with single mutations at Leu315 and

Leu444 when the cysteines are reduced. The greatest increase

of all (412 fold) came with the conformation-restricting

linker mutation L320P (Figure 7C).

Discussion

HKs are multifunctional enzymes that participate in auto-

kinase, phosphotransferase and phosphatase reactions, and

their phospho-accepting histidines typically have roles in

all three activities (Hsing et al, 1998). The kinase and/or

phosphatase activities are regulated by sensor input and are

dependent on the presence of nucleotide (reviewed in Stock

et al, 2000). These observations, together with the large

separation between ATP and the phosphoacceptor histidine

in this structure of HK853-CD, suggest that the cytoplasmic

K383

Y384

D411

R430

HK853

ADPββN

K392

Y393

D415

R439

Q442R434

N389

N385

PhoQ

AMPPNP

Figure 5 Comparison of the nucleotide-binding site of the TM0853and PhoQ HKs. Secondary structures surrounding the ATP-bindingsite are drawn as gray ribbons and the ATP lids are in magenta. Thenucleotides and the residues interacting with their phosphates aredepicted as sticks and labeled. The Mg2þ ion of PhoQ is drawn as acyan sphere. Hydrogen bonds are shown as dotted lines. Electrondensity of the HK853 nucleotide is contoured as a semitransparentblue surface at a level of 1s.



portions of HK sensor must access multiple conformational

states, some of which are critical for catalytic action.

We present a structure-based scheme for the multiple

activities of HKs in Figure 8. This scheme is based on the

structure in the state found here (Figure 3) and on models for

other relevant states (Figure 9). In unphosphorylated state A,

a kinase domain loaded with ATP is poised to phosphorylate

the acceptor histidine. Upon sensor input, if needed, this

kinase domain is freed to adopt a conformation appropriate

for forming the A–B transition state with the phosphoaccep-

tor histidine on the opposite protomer. In phosphorylated

state B, the kinase domain is relaxed to a conformation

whereby the site surrounding the phosphorylated histidine

is available for interaction in transition state B–A* with a

cognate RR. Phosphotransfer to the acceptor aspartyl residue

can then ensue. In unphosphorylated state A*, which may

depend on sensor input, the interdomain conformation

permits interaction with a cognate phosphorylated regulatory

domain in the A*–A intermediate, and this accelerates aspar-

tate dephosphorylation.

The scheme depicted in Figure 8 has symmetric ground

states and asymmetric intermediate structures, but our mod-

eling (Figure 9) is also consistent with symmetric inter-

mediates where both phospho-accepting histidines are

equivalently engaged. Asymmetry is observed in the auto-

phosphorylation and phosphatase reactions of NtrB (Jiang

et al, 2000; Pioszak and Ninfa, 2003), but both EnvZ–OmpR

and FixL–FixJ form 2:2 HK–RR phosphotransfer complexes

(Miyatake et al, 1999; Yoshida et al, 2002).

Implications of the HK853-CD structure for catalysis

and regulation

Our crystal structure of HK853-CD has attributes appropriate

for a model of class I HK sensors in all ground states (A, B

and A*) and for intermediates in the phosphotransfer and

phosphatase reactions. It is, however, obviously inappropri-

ate for the autokinase reaction since the imidazole of His260

and the b-phosphate position of the nucleotide are separated

by 25 A and wrongly oriented for interaction. How then might

this crystal structure relate to the states depicted in the cycle

of catalytic reactions depicted in Figure 8? Two features of the

structure seem relevant in this regard. First, although this

is an unphosphorylated protein molecule, we propose that

the ordered sulfate ion near the phosphorylatable histidine

mimics the phosphate group in the phosphohistididyl pro-

tein. This sulfate ion is hydrogen bonded to the Ne atom of

His260 and also to side chains of conservative Arg3170 and

other groups from the opposing protomer. In that sense the

structure may represent a model for ground state B, which

catalyzes phosphotransfer to the RR.

The second structural feature relevant to the reaction

scheme is concerned with the interdomain contacts. The

C-terminal half of helix a2 is a central participant in this

interaction, and the corresponding sequence is part of the

weakly conserved X motif identified in a mutational analysis

of EnvZ (Hsing et al, 1998). Most mutations that eliminate

phosphatase activity without diminishing kinase activity

(KþP�) mapped to this region. The position of Tyr287

in EnvZ (Leu315 in HK853) seems especially critical since it

was affected in multiple isolates from the mutational screen.

Leu315 has a central place in the interdomain interface

(Figure 6), making contacts with catalytic domain residues

Leu444, Ala447 and Ile448, consistent with a pivotal role in

stabilizing phosphatase state A*. Mutations at nearby EnvZ

residues, including Arg289 (sulfate ligand Arg317 in HK853),

also confer a KþP� phenotype. The X motif mutation L288P

had no effect on the residual phosphatase activity of the

isolated DHp domain from EnvZ (Zhu et al, 2000), however,

consistent with the dependence of regulated phos-

phatase action on interdomain contacts. Key residues from

ADP ADP

M250 M250

I247 I247

F254 F254

F312 F312

K251 K251

αα1 α1

α2 α2L315 L315

D311 D311L320 L320

I322 I322

L455 L455R369 R369

E451 E451I452 I452

Q372 Q372

A447 A447L444 L444

F428 F428

Q427 Q427

I448 I448

α1′ α1′

SO4–2 SO4

–2

R317 R317

R314 R314

S319 S319

H260′ H260′

Figure 6 Interactions between DHp and CA domains. Stereoview of the structural elements involved in interdomain contacts and sulfate ioninteractions. The DHp domain, CA domain and interdomain-connecting loop are represented in blue, gold and green ribbon diagrams,respectively. Additionally, the a10 helix, which presents His2600 as a sulfate ligand, is shown in gray. The interacting side chains are shown assticks with the same carbon atom color as the corresponding domain, except the sulfate-interacting residues that are in gray. Nitrogen, oxygen,sulfur and nucleotide molecule are drawn in blue, red, black and magenta, respectively. Residue labels take the colors of their domains.Hydrogen bonds and salt bridges between the sulfate ion and interacting residues are indicated by purple dots.



the catalytic-domain side of the interface, including Phe428

and Leu444, are near the nucleotide within the ATP-lid

segment (Figure 6), consistent with NtrB mutations

(Pioszak and Ninfa, 2003) and the dependence of phospha-

tase activity on the presence of nucleotide (Keener and Kustu,

1988; Jung and Altendorf, 1998).

We expect that regulatory signals act to control interfacial

stability. Signals from the external sensor domain, presum-

ably transduced through the coiled-coil segment to the

four-helix bundle, must ultimately affect the viability of the

interface, destabilizing it for kinase action and stabilizing it

for phosphatase action. This interface clearly must give way

to permit the catalytic domain to swing around and perform

the trans-histidine phosphorylation. Indeed, our mutational

disruptions of the interface do accelerate autokinase activity,

whereas activity is blocked upon interface stabilization.

Signal-dependent conformational changes within the four-

helix bundle may further distinguish states A, B and A*.

Model for the autophosphorylation reaction

of HK853-CD

Since the conformation of HK853-CD in our crystals is

inappropriate for the autokinase reaction, we have modeled

the disposition of domains needed for the autophosphory-

lation reaction. This was done by docking an isolated cata-

lytic domain (residues 322–489) onto dimeric DHp domains

(residues 232–317) in a manner consistent with phospho-

transfer from ATP to histidine, and then considering con-

straints imposed in reconnecting the linker peptide (residues

317–322). ATP was modeled into the catalytic domain by

adding g-phosphate to ADPbN as in the AMPPNP of CheA

(Bilwes et al, 2001), and the Ob1–Pg bond of this ATP was

aligned with the Cb–Cg bond of His260 in the DHp domain

with the catalytic domain, separated such that the Ob1(ATP)–

Ne2(His260) distance would be appropriate for the kinase

transition state (B4.5 A). Such models were constructed

for His260 in each of the three major conformations for this

residue, m, t and p (w1 at minus 601 (49% abundance), trans

(32%) and plus 601 (13%) following Lovell et al, 2000), and

for rigid-body rotations at 101 intervals about the axis defined

by Ob1(ATP)–Cb(His260). Each docking was tested for

cis and trans connectivity between domains.

All models generated in the m histidine conformation of

the crystal structure have serious steric clashes or present

infeasible linkages. Models without steric problems can be

generated in the p conformation for both cis- and trans-

autophosphorylation reactions, but this conformation is un-

favored. His260 is most exposed when in the t conformation,

which is as observed in the Spo0B–Spo0F complex (Zapf

et al, 2000). A small range of t models can be connected in

favorable linker peptide conformations for the trans mode

of reaction. One of these, presenting optimal shape comp-

lementarity free of steric conflicts, is shown in Figure 9.

Conformational changes to achieve this model are in linker

residues 317–320 and, in keeping with this, the conformation-

restricted mutant L320P is hyperactive.

Model for the interaction of HK853-CD with its RR

In the current absence of structural information on complexes

between DHp and RR domains, we have modeled this inter-

action for HK853 by analogy with the cognate complex

between Spo0B and Spo0F, phosphorelay components for

sporulation in B. subtilis (Zapf et al, 2000). Spo0B is evolu-

tionarily and structurally (Figure 4) related to HKs, and it can

freely transfer a phosphoryl group to or from the intermediate

RR Spo0F. It does this via a histidine residue that corresponds

to the phosphoacceptor histidine in the kinase homologs, and

this residue is in proximity with the phosphorylatable aspar-

tate in the Spo0B–Spo0F complex. We constructed an HK853–

RR complex by superimposing the phospho-accepting

histidine helices of Spo0B and HK853–CD, thereby orienting

Figure 7 Autokinase activity of interfacial mutant variants.(A) Mutations at the interfacial residue Ile448. (B) Double-cysteinemutations when reduced by DTT (open symbols) and when oxi-dized (filled symbols), where the negligible activity is overlapping.(C) Mutations to proline in the linker segment.



Spo0F into a hypothetical phosphotransfer complex with

HK853 (Figure 9).

This Spo0B–Spo0F-based model has properties appropriate

for the HK853–RR complex. HK853–CD readily accommo-

dates Spo0F with only minor clashes and with burial of

substantial surface into the interface (2250 A2 in total). The

most significant steric clash is between the sensitive X region

of HK853–CD, discussed above, and the loop between b4 and

a4 of Spo0F (Figure 9C), which undergoes structural changes

upon RR activation (Birck et al, 1999; Lewis et al, 1999). The

surface of HK853–CD that is buried into the phosphotransfer

interface covers much of helix a1b downstream of the

phospho-accepting histidine (Figure 9B), consistent with

NMR titration experiments of the OmpR receiver domain

interacting with the isolated EnvZ DHp domain (Tomomori

et al, 1999). Finally, although His260 is poorly oriented for

phosphotransfer in its HK853-CD m conformation, a produc-

tive t conformation similar to that observed in the Spo0B–

Spo0F complex (Zapf et al, 2000) is achieved by a simple v1

rotation, whereby Ne of His260 comes to lie an appropriate

5.1 A from an Od of Asp54 in Spo0F.

Implications of the coiled-coil region for signal

transduction

The coiled-coil portion of HK853-CD (residues 232–253

of helix a1a) extends into the DHp domain and we expect it

to emerge directly from the transmembrane, four-helix bun-

dle of this sensor protein. It must thereby play a role in signal

transduction. Comparable coiled-coil segments are predicted

to exist in this and other HK sensors (Figure 1); indeed, Singh

et al (1998) identified putative coiled-coil helical structures

preceding the phospho-accepting histidine in 76% of 189

class I HKs. The HAMP domain linkers that typically connect

the transmembrane domains of these and other sensors to

their catalytic domains have been predicted to consist of two

amphipathic helices separated by a loop region (Butler and

Falke, 1998; Williams and Stewart, 1999). The linker segment

sometimes includes whole additional domains, such as the

cysteine-cluster domain of NarX (Stewart, 2003) and the PAS

domain of DcuS (Golby et al, 1999). Typically, the second

HAMP helix corresponds to the coiled-coil segment found

in HK853–CD and predicted for most others. The HAMP

segment of chemotactic receptors is stably folded (Butler and

Falke, 1998) and has been modeled as a continuous coiled coil

that also extends through the membrane and into the periplas-

mic sensor domain (Kim et al, 1999; Falke and Hazelbauer,

2001). This is analogous to the topology in HK853 and may

be typical, perhaps with extra loops and domains bulging as

gall-like extrusions from the coiled-coil stem.

The coiled coil in HK853–CD has hydrophilic residues

occupying several of the normally hydrophobic a and d

contact positions of the canonical heptad repeats (Figures 1

N N′

ATPATP H

N N′

C’

N′

ATPH*

C′

D*

ATPC′

N′D

D*

N N′

ATPH

C′

ATP

N′

C′

A B

A*

D

N′

N

N

Figure 8 Structure-based schematic of the reactions catalyzed by HK sensors. The kinase autophosphorylation (A-B), phosphotransferase(B-A*) and phosphatase (A*-A) activities are shown on projected outlines of the enzyme and protein–substrate models. Positions of N andC termini, ATP and the phospho-accepting histidine (H) are indicated on an HK dimer (orange and green). Position of phospho-acceptingaspartate (D) is indicated on a RR (red). The transferred phosphoryl group is indicated as a yellow asterisk.



and 4B), and the predicted coiled coils in related sensor

proteins also show a weak hydrophobic character of the

a–d core (Tao et al, 2002). This property may confer an

interfacial plasticity of importance in signal transmission.

Mutations that increase the hydrophobicity of the predicted

coiled-coil core tend to bias various HKs toward one or the

other signaling state (Kalman and Gunsalus, 1990; Tokishita

et al, 1992; Tao et al, 2002). Included among these are

substitutions and short deletions in the HAMP domain of

EnvZ that alter the ratio of kinase-to-phosphatase activity

(Park and Inouye, 1997).

Signal transduction by class I HK sensors begins with

changes in the sensor domains induced by ligands or other

stimuli. Conformational changes are transduced through

the transmembrane four-helix bundle into the cytoplasmic

domain of the dimeric receptor. These changes ultimately

affect the kinase and/or phosphatase activities mediated by

the catalytic domains. Two transduction models have been

proposed: (i) a rotational movement of the helices with

respect to one another (Cochran and Kim, 1996) and (ii)

a piston-like movement of one or two helices with respect to

the other helices in the bundle (reviewed by Falke and

Hazelbauer, 2001), favored by the preponderance of evidence

from chemotactic receptors. The coiled-coil linker domains

may serve to modulate and perhaps amplify these move-

ments, but in any case they must transmit the signal. It is

apparent from the structure and mutational analysis of

HK853–CD that even subtle changes could affect the latch

between the helical-hairpin and kinase domains and the

disposition of the phospho-accepting histidine residue.

Just as signals transduced through coiled coils from across

the membrane may effect these changes in class I kinases,

regulatory factors that modulate other kinases may act on

comparable interfaces.

Materials and methods

Cloning and protein productionORF TM0853 was cloned from genomic T. maritima DNA forrecombinant expression in E. coli. Vectors were designed to producethe putative sensor HK, both histidine-tagged in full length forlocalization assays (His-HK853), and as the predicted cytoplasmicdomain (residues 232–489) for structure analysis (HK853–CD).Plasmid pHK853 was constructed by cloning TM803 into a pBR322-derived plasmid designed for in vivo phosphotransfer assays(Regelmann et al, 2002). Mutant variants of HK853–CD weredesigned for a battery of functional tests and to add methionineresidues for Se MAD phasing. HK853–CD was purified byammonium sulfate fractionation, ion-exchange chromatographyand size exclusion chromatography. Predicted size and complete Seincorporation were confirmed by mass spectrometry.

Cellular and biochemical characterizationCellular localization of the full-length sensor kinase was deter-mined, as a function of temperature, by ultracentrifugal separationof soluble and membrane fractions followed by pulldown on His-HK853 on Ni-chelating beads and staining on SDS–PAGE gels.Autokinase activity was assayed, as described before (Marina et al,2001), by following the incorporation of radiolabel from [g-32P]ATPinto purified wild-type or mutant HK853–CD as separated on SDS–PAGE gels. Phosphotransfer to PhoP or OmpR in vivo was assayedby measuring b-galactosidase activity from E. coli strains harboringPhoP-activated phoN-lacZ (Waldburger and Sauer, 1996) or OmpR-activated ompC-lacZ (Hsing and Silhavy, 1997) fusions, respec-tively, after transformation by pHK853 or control plasmids.

90°Spo0B:Spo0F HK853:Spo0F

A B C

ED

Figure 9 Models of complexes for the phosphotransferase and kinase reactions catalyzed by HK853-CD. (A) Ribbon representation ofthe experimental complex (Zapf et al, 2000) between Spo0B (green and yellow) and Spo0F (red). For clarity, only one Spo0F molecule is drawn.(B) The Spo0F RR (red) docked onto the HK853–CD dimer (green and yellow) as a model of the phosphotransferase complex (see text). Thecatalytic histidine and aspartate residues and the nucleotide are shown as stick models. (C) Orthogonal view of (B). (D) Model of HK853–CDpoised for the autokinase reaction. The catalytic domain of one protomer has been moved to align the g phosphate of its ATP moiety with thephosphoaccepting histidine of the other promoter to permint trans-phosphorylation. The histidine and nucleotide are shown in stickrepresentation. (E) Orthogonal view of (D).



Nucleotides were identified by FPLC separations from washedcrystals or from nucleotide hydrolysis reactions in comparison withretention times of various adenosine nucleotides.

Crystallographic analysisCrystals were grown from 1.25 M Li2SO4 and 50–200 mM ammo-nium acetate at pH 6.5. They are in space group C2221 with unit celldimensions a¼ 79.3 A, b¼ 162.1 A and c¼ 42.5 A. Cryopreservationwas achieved in mother liquor plus 7.5% ethylene glycol and 15%sucrose. The structure was solved at 2.1 A resolution from a MADexperiment based on selenomethionyl I370M/V373M HK853–CDand refined at 1.9 A resolution against wild-type native HK853–CD.All diffraction data were measured at NSLS beamline X4A. Resultsare deposited with PDB accession code 2C2A.

Supplementary dataSupplementary data are available at The EMBO Journal Online.

Acknowledgements

We thank C Mott for assistance in plasmid production, J Escolanoand I Esmorıs for help in purifying mutant proteins, and members ofthe Hendrickson and Waldburger laboratories for helpful discus-sions. This work was supported in part by NIH grants GM34102(WAH) and AI41566 (CDW), and by Ministerio de Ciencia yTecnologıa grant BIO2002-03709 (AM) in Spain. Beamline X4A atthe National Synchrotron Light Source (NSLS), a DOE facility, issupported by the New York Structural Biology Center.

References

Aravind L, Ponting CP (1999) The cytoplasmic helical linker domainof receptor histidine kinase and methyl-accepting proteins iscommon to many prokaryotic signalling proteins. FEMSMicrobiol Lett 176: 111–116

Bilwes AM, Alex LA, Crane BR, Simon MI (1999) Structure of CheA,a signal-transducing histidine kinase. Cell 96: 131–141

Bilwes AM, Quezada CM, Croal LR, Crane BR, Simon MI (2001)Nucleotide binding by the histidine kinase CheA. Nat Struct Biol8: 353–360

Birck C, Mourey L, Gouet P, Fabry B, Schumacher J, Rousseau P,Kahn D, Samama JP (1999) Conformational changes induced byphosphorylation of the FixJ receiver domain. Struct Fold Des 7:1505–1515

Butler SL, Falke JJ (1998) Cysteine and disulfite scanning revealstwo amphiphilic helices in the linker region of the aspartatechemoreceptor. Biochemistry 37: 10746–10756

Chou K, Maggiora GM, Nemethy G, Scheraga HA (1988) Energeticsof the structure of the four-a-helix bundle in proteins. Proc NatlAcad Sci USA 85: 4295–4299

Cochran AG, Kim PS (1996) Imitation of Escherichia coli aspartatereceptor signaling in engineered dimers of the cytoplasmicdomain. Science 271: 1113–1116

Cserzo M, Wallin E, Simon I, von Heijne G, Elofsson A (1997)Prediction of transmembrane alpha-helices in procariotic mem-brane proteins: the Dense Alignment Surface method. Prot Eng10: 673–676

Dutta R, Inouye M (2000) GHKL, an emergent ATPase/kinasesuperfamily. Trends Biochem Sci 25: 24–28

Dutta R, Qin L, Inouye M (1999) Histidine kinase: diversity ofdomain organization. Mol Microbiol 34: 633–640

Fabret C, Feher VA, Hoch JA (1999) Two-component signal trans-duction in Bacillus subtilis: how one organism sees its world.J Bacteriol 181: 1975–1983

Falke JJ, Hazelbauer GL (2001) Transmembrane signaling in bacter-ial chemoreceptors. Trends Biochem Sci 26: 257–265

Golby P, Davies S, Kelly DJ, Guest JR, Andrews SC (1999)Identification and characterization of a two-component sensor-kinase and response-regulator system (DcuS–DcuR) controllinggene expression in response to C-4-dicarboxylates in Escherichiacoli. J Bacteriol 181: 1238–1248

Hsing W, Russo FD, Bernd KK, Silhavy TJ (1998) Mutations thatalter the kinase and phosphatase activities of the two-componentsensor EnvZ. J Bacteriol 180: 4538–4546

Hsing W, Silhavy TJ (1997) Function of conserved histidine-243 inphosphatase activity of EnvZ, the sensor of porin osmoregulationin Escherichia coli. J Bacteriol 179: 3729–3735

Hultquist DE, Moyer RW, Boyer PD (1996) The preparation andcharacterization of 1-phosphohistidine and 3-phosphohistidine.Biochemistry 5: 322–331

Jiang P, Peliska JA, Ninfa AJ (2000) Asymmetry in the autopho-sphorylation of the two-component regulatory system transmitterprotein nitrogen regulator II of Escherichia coli. Biochemistry 39:5057–5065

Jung K, Altendorf K (1998) Truncation of amino acids 12–128causes deregulation of the phosphatase activity of thesensor kinase KdpD of Escherichia coli. J Biol Chem 273:17406–17410

Kalman LV, Gunsalus RP (1990) Nitrate-independent and molybde-num-independent signal transducction mutations in narX thatalter regulation of anaerobic respiratory genes in Escherichia coli.J Bacteriol 172: 7049–7056

Keener J, Kustu S (1988) Protein kinase and phosphoproteinphosphatase activities of nitrogen regulatory proteins NTRB andNTRC of enteric bacteria: roles of the conserved amino-terminaldomain of NTRC. Proc Natl Acad Sci USA 85: 4976–4980

Kim KK, Yokota H, Kim SH (1999) Four-helical-bundle structure ofthe cytoplasmic domain of a serine chemotaxis receptor. Nature400: 787–792

Lewis RJ, Barnnigan JA, Muchova K, Barak I, Wilkinson AJ (1999)Phosphorylated aspartate in the structure of a response regulatorprotein. J Mol Biol 294: 9–15

Lovell SC, Word JM, Richardson JS, Richardson DC. (2000) Thepenultimate rotamer library. Proteins 40: 389–408

Marina A, Mott C, Auyzenber A, Hendrickson WA, Waldburger CD(2001) Structural and mutational analysis of PhoQ histidinekinase catalytic domain. J Biol Chem 276: 41182–41190

Miyatake H, Mukai M, Adachi S, Nakamura H, Tamura K, Iizuka T,Shiro Y, Strange RW, Hasnain SS (1999) Iron coordination struc-tures of oxygen sensor FixL characterized by Fe K-edge extendedx-ray absorption fine structure and resonance Raman spectro-scopy. J Biol Chem 274: 23176–23184

Ninfa EG, Atkinson MR, Kamberov ES, Ninfa AJ (1993) Mechanismof autophosphorylation of Escherichia coli nitrogen regulator II(NRII or NtrB): trans-phosphorylation between subunits.J Bacteriol 175: 7024–7032

Park H, Inouye M (1997) Mutational analysis of the linker region ofthe EnvZ, an osmosensor in Escherichia coli. J Bacteriol 179:4382–4390

Parkinson JS, Kofoid EC (1992) Communication modules in bacter-ial signaling proteins. Annu Rev Genet 26: 71–112

Pioszak AA, Ninfa AJ (2003) Mechanism of the PII-activatedphosphatase activity of Escherichia coli. NRII (NtrB): how thedifferent domains of NRII collaborate to act as a phosphatase.Biochemistry 42: 8885–8899

Qin L, Dutta R, Kurokawa H, Ikura M, Inouye M (2000) A mono-meric histidine kinase derived from EnvZ, an Escherichia coliosmosensor. Mol Microbiol 36: 24–32

Regelmann AG, Lesley JA, Mott C, Stokes L, Waldburger CD (2002)Mutational analysis of the Escherichia coli PhoQ sensor kinase:differences with the Salmonella enterica serovar TyphimuriumPhoQ protein and in the mechanism of Mg2+ and Ca2+ sensing.J Bacteriol 184: 5468–5478

Russo FD, Silhavy TJ (1993) The essential tension: opposed reac-tions in bacterial two-component regulatory systems. TrendsMicrobiol 1: 306–310

Singh M, Berger B, Kim PS, Berger J, Cochran A (1998)Computational learning reveals coiled coil-like motifs inhistidine kinase linker domains. Proc Natl Acad Sci USA 95:2738–2743

Song Y, Peisach D, Pioszak AA, Xu Z, Ninfa AJ (2004) Crystalstructure of the C-terminal domain of the two-component systemtransmitter protein nitrogen regulator II (NRII; NtrB), regulatorof nitrogen assimilation in Escherichia coli. Biochemistry 43:6670–6678



Stewart V (2003) Nitrate- and nitrite-responsive sensors NarX andNarQ of proteobacteria. Biochem Soc Trans 31: 1–10

Stock AM, Robinson VL, Goudreau PN (2000) Two componentsignal transduction. Annu Rev Biochem 69: 183–215

Tanaka T, Kawata M, Mukai K (1991) Altered phosphorylation ofBacillus subtilis DegU caused by single amino acid changes inDegS. J Bacteriol 173: 5507–5515

Tanaka T, Saha SK, Tomomori C, Ishima R, Liu D, Tong KI, Park H,Dutta R, Swindells MB, Yamazaki T, Ono AM, Kainosho M,Inouye M, Ikura M (1998) NMR structure of the histidine kinasedomain of the E. coli osmosensor EnvZ. Nature 396: 88–92

Tao W, Malone CL, Ault AD, Deschenes RJ, Fassler JS (2002)A cytoplasmic coiled-coil domain is required for histidinekinase activity of the yeast osmosensor, SLN1. Mol Microbiol43: 459–473

Tokishita S, Kojima A, Mizuno T (1992) Transmembranes signaltranduction and osmoregulation in Escherichia coli: functionalimportance of the transmembrane regions of membrane-locatedprotein kinase EnvZ. J Biochem 111: 707–713

Tomomori C, Tanaka T, Dutta R, Park H, Saha SK, Zhu Y, Ishima R,Liu D, Tong KI, Kurokawa H, Qian H, Inouye M, Ikura M(1999) Solution structure of the homodimeric core domainof Escherichia coli histidine kinase EnvZ. Nat Struct Biol 6:729–734

Varughese KI, Madhusudan Zhou XZ, Whiteley JM, Hoch JA (1998)Formation of a novel four-helix bundle and molecular recognitionsites by dimerization of a response regulator phosphotransferase.Mol Cell 2: 485–493

Waldburger CD, Sauer RT (1996) Signal detection by the PhoQsensor-transmitter. Characterization of the sensor domain and aresponse-impaired mutant that identifies ligand-binding determi-nants. J Biol Chem 271: 26630–26636

Williams SB, Stewart V (1999) Functional similarities among two-component sensors and methyl-accepting chemotaxis proteinssuggest a role for linker region amphipathic helices in transmem-brane signal transduction. Mol Microbiol 33: 1093–1102

Yang Y, Inouye M (1991) Intermolecular complementation betweentwo defective mutant signal-transducing receptors of Escherichiacoli. Proc Natl Acad Sci USA 88: 11057–11061

Yoshida T, Qin L, Inouye M (2002) Formation of the stoichiometriccomplex of EnvZ, a histidine kinase, with its response regulator,OmpR. Mol Microbiol 46: 1273–1282

Yount RG, Babcock D, Ballantyne W, Ojala D (1971) Adenylylimidodiphosphate, an adenosine triphosphate analog containinga P–N–P linkage. Biochemistry 10: 2484–2489

Zapf J, Sen U, Madhusudan, Hoch JA, Varughese KI (2000) Atransient interaction between two phosphorelay proteinstrapped in a crystal lattice reveals the mechanism of molecularrecognition and phosphotransfer in signal transduction. Structure8: 851–862

Zhou H, Dahlquist FW (1997) Phosphotransfer site of the chemo-taxis-specific protein kinase CheA as revealed by NMR.Biochemistry 36: 699–710

Zhu Y, Qin L, Yoshida T, Inouye M (2000) Phosphatase activity ofhistidine kinase EnvZ without kinase catalytic domain. Proc NatlAcad Sci USA 97: 7808–7813



JOURNAL Structure of the entire cytoplasmic portion of a...

Documents

Transcript of JOURNAL Structure of the entire cytoplasmic portion of a...